The Function And Expression Regulation Of MiR-196B And MiR-30a In Chronic Myeloid Leukemogenesis

Chronic Myelogenous Leukemia, referred to as CML, is malignant myeloproliferative disorders in the hematopoietic stem cells and the most common type of chronic leukemia.95%of CML has Ph chromosome-specific t (9;22)(q34; qll) karyotype and the expression of BCR-ABL fusion gene. The Ph chromosome is the result of translocation of the long arm of chromosome9and22, The fusion gene BCR-ABL1was formed from the translocation of the long arm of chromosome9(9q34) on the proto-oncogene ABL and chromosome22(the22q11) in the BCR gene. Different BCR-ABL1fusion gene type was associated with the disease phenotype. In BCR-ABL1fusion gene, malignant information is mainly carried by the ABL segment, ABL1is a non-receptor tyrosine kinase of the Abelson (leukemia virus) family and currently mainly used in the malignant tumor, while breakpoints in the BCR mainly affect the disease phenotype. In CML, the BCR-ABL1fusion gene transcripts to the8.5kb mRNA, and translated to produce the molecular mass for a210KD BCR-ABL1fusion protein P210BCR/ABL, the protein with abnormal tyrosine kinase activity with the ABL SH1structure abnormal domain TK activity, resulting in ATP binding, combined with the ABL signal transduction protein phosphorylation and activation, and effect on a series of downstream signaling pathways, including the RAS-MAPK pathway, the JAK-STAT pathway, the PI-3K kinase pathway, MYC pathway, not only interfere with cytokine proliferation and inhibit apoptosis, but also caused the hematopoietic stem cells to regulate growth and differentiation of the lack of adhesion procedures, eventually leading to a large number of bone marrow progenitor cells proliferation, apoptosis, reduction of bone marrow stromal cells adhesion decreased, so that a large number of immature myeloid cells released into the peripheral blood, leading to the occurrence of chronic myeloid leukemia. Therefore, BCR-ABL1fusion gene is considered to be chronic myeloid leukemogenesis of the molecular pathological basis and also an effective indicator of CML diagnosis, observation, prognosis, and monitoring. Recent studies about BCR-ABL1focused on the tyrosine kinase inhibitors and signal transduction pathways, and miRNA-related researches are rarely.miRNAs are a class of small non-coding single-stranded RNA,about21-25nt nucleus in length,which are processed from the precursor to form a hairpin structure about70nt size. miRNAs regulate post-transcriptional gene expression by degrading target mRNA or inhibiting their translation through complementary base pairing of the target mRNA. miRNAs expression are organized and time-specific. miRNAs are important regulatory molecules of other functional gene expression involving in cell proliferation, apoptosis, differentiation, metabolism, growth, tumor metastasis and a variety of other biological processes. miRNA expression is associated with various tumors, about50%of annotated miRNAs located at tumor-associated fragile sites in the genome, indicating that miRNA play a critical role in tumorigenesis. The study of miRNAs on tumor formation indicates miRNAs not only inhibit tumorigenesis, but also promote carcinogenesis.Declining or losing of miRNA expression which act as tumor suppressor gene may be caused by any step of miRNA biological synthesis defects, which will lead to the inappropriate expression of miRNA target protein, eventually could result in excessive cell proliferation, invasion, apoptosis reduction and abnormal differentiation, causing tumor formation finally. miRNA overexpression of oncogene function will also lead to tumorigenesis, in this case, due to the amplification of the miRNA genes and persistent promoter activity increasing the efficiency of miRNA processing, or improving the stability of the miRNA, which will result in increasing expression of these miRNA in abnormal tissue or at inappropriate developmental stage, the expression of its target genes (tumor suppressor gene) expression was decreased and cause the tumor finally.In this study, we firstly used several bioinformatics prediction software to predicte miRNAs of the BCR-ABL1gene, relative prediction software included TargetScan, PicTa, the miRBase, miRanda, Mirnaviewer and DIANA TarBase. The results showed that miRNA targeting BCR-ABL1included miR-196b and miR-30a; following by forecasting the target gene of miR-196b and miR-30a in CML throught bioinformatics software. We integrated the results of various software predictions, indicating that the target genes of miR-196b may include BCR-ABL1and HOXA9, target genes of miR-30a may include BCR-ABL1, this study was intended to target miR-196b and miR-30a as the research object, and verified their function and expression regulation in CML.Firstly, we used total RNA of K562cell as a template, amplifying the BCR-ABL1-mRNA3’UTR sequence and HOXA9mRNA3’UTR sequence, then connected with psiCHECK TM-2vector to construct the recombinant plasmid psiCHECK TM-2-BCR-ABL1-3’UTR and psiCHECK TM-2-HOXA9-3’UTR. The above recombinant plasmids were used as templates and proceeded the subclone. The method of SOE was adopted to proceed mutation of seed sequence of miR-196b, miR-30a binding sites in BCR-ABL1-3’UTR and HOXA9-3’UTR to build the recombinant plasmids psiCHECK TM-2-the BCR-ABLl-3’UTR-Mutant-196b, psiCHECK TM-2-BCR-ABL1-3’UTR-Mutant-30a, psiCHECK TM-2-HOXA9-3’UTR-Mutant. Secondly, we used the dual luciferase reporter gene assay system to confirm the target gene. Mimics group, NC group, mutant group and the mock group were established. the transfection experiments were carried out in293T cells, then we detected fluorescence density after48h, results were analysed by One Way ANOVA of SPSS13.0. The data showed that there are significant differences (P <0.05) between196b mimics+ABL1,196b mimics reduced the fluorescence value, and there are no significant differences (P>0.05) between four other control groups, indicated the target gene of miR-196b including the BCR-ABL1. There are significant differences (P<0.05) between196b mimics+HOXA9,196b mimics reduced the fluorescence value, and there are no significant differences (P>0.05) between four other control groups, indicated that the miR-196b target genes including HOXA9. There are significant differences (P<0.05) between30a mimics+ABL1,30a mimics reduced fluorescence value, and there are no significant differences (P>0.05) between four other control groups, indicated that miR-30a target genes including BCR-ABL1.In order to further verify the correspondent relation between miRNA and its target genes, functional studies of miR-196b and miR-30were carried out. First of all, human genomic DNA was used as templates, we amplificated miR-196b precursor pre-miR-196b and miR-30a precursor pre-miR-30a, and connected both precursors to vector plVTHM, constructing lentiviral expression vector plVTHM-miR-196b and plVTHM-miR-30a. We co-transfected lentiviral expression vector and plasmid psPAX2, pMD2.G into293T cells, packaged virus, and then infected K562cells with virus solution, constructing overexpression of miR-196b cells and overexpression of miR-30a cells. Then we studied cell proliferation, cell cycle, miRNA expression and the expression of target gene BCR-ABL1and HOXA9 in overexpression of miRNA cells. Second, we transfected miRNA inhibitor into overexpression of miRNA cells to lower miRNA expression in the cells, then cell proliferation, cycle and the expression of target gene BCR-ABL1and HOXA9were measured to identified whether the cells function could restore. Finally, the target genes BCR-ABL1and HOXA9were transfected with the corresponding siRNA, and we detected cell proliferation, cell cycle and the expression of target genes BCR-ABLl and HOXA9to determine whether the inhibition effects were conform to overexpression of miRNA cells. The results showed that overexpression of miR-196b in K562cells inhibited cell proliferation significantly, G2retardation, the BCR-ABL1protein and HOXA9protein were decreased. Inhibiting the expression of miR-196b in miR-196b overexpression cells would enhance cell proliferation, restoring cell cycle, the levels of BCR-ABLl protein and HOXA9protein were elevated. Overexpression of the miR-30a in K562cells inhibited cell proliferation significantly, arresting in the G1phase, and the levels of BCR-ABLl protein was decreased. Inhibiting the expression of miR-30a in miR-30a overexpression cells would enhanced proliferation, restoring cell cycle, the BCR-ABL1protein level significantly increased. After transfection of ABL1siRNA into K562cells, BCR-ABLl mRNA levels and protein expression levels of BCR-ABL1decreased, cell proliferation was inhibited, arresting in G1phase, proliferation rate was basically the same with overexpression of miR-30a cells. After transfection of K562cells with HOXA9siRNA, HOXA9mRNA levels and HOXA9protein expression levels decreased, cell proliferation inhibited, arresting in G1phase and faster than overexpression of miR-196b cells. Gaining function and losing function study in cells further proved:target genes of miR-196b included BCR-ABL1and HOXA9and target genes of included HOXA9, both miR-196b and miR-30a played a tumor inhibition role in CML. miRNA expression is often related with epigenetic, epigenetic refers to the DNA sequence remains stable but gene expression has undergone a heritable change, it is induced by other genetic material other than genetic information from cells.and could be stably delivered in cell development and proliferation process There are many different types of epigenetic phenomena, but the epigenetic phenomenon of DNA methylation and histone modifications are the most common forms and are closely associated with malignancy development, especially CpG island methylation of tumor suppressor gene could induce transcriptional inactivation and post-translational modifications of N terminal domain DNA methylation and histone modifications become an important branch of epigenetic in the field of cancer research. DNA methylation includes the entire genome demethylation and part of the regional hypermethylation, and the role of DNA methylation in tumor development is particularly evident. Histone modification is also one of the epigenetic mechanisms of the gene expression regulation and tumorigenesis. Many studies have revealed that miRNAs are regulated by epigenetic regulation of methylation and histone modifications. Epigenetic regulates miRNA molecules and directly involves in the process of tumor development. Therefore, the study would be to investigate epigenetic regulation of miR-196b and miR-30a in CMLFirst, we used the demethylation drug5-Aza-2’-dc to treat K562cells, and then detected the cell cycle, apoptosis, expression of miR-196b and miR-30a, BCR-ABL1and HOXA9expression in K562cells before and after dealing with5-Aza-2’-dc Second, promoter CpG island of miR-196b and miR-30a were predicted and the BSP was proceeded to detect promoter CpG island methylation levels of miR-196b in K562cells before and after5-Aza-2’-dc treatment, We elected a series of relatively high degree of methylation and relatively large changes sequence before and after treatment according to the BSP results, and detected in clinical samples. Finally, total RNA of16cases of chronic myeloid leukemia patients and10normal persons were extracted to detect expression levels of miR-196b and miR-30a,45patients with leukemia and10cases of normal human genome were extracted.We used the MSP assay to detect CpG island methylation levels of miR-196b promoter. Results of MSP were statistically analyzed using multi-sample rate χ2of SPSS13.0. The results indicated that proliferation rate was inhibited, arresting in G2phase and undergoing apoptosis in K562cells treated with5-Aza-2’-dc. In K562cells, miR-196b promoter has relatively high degree of methylation, after treating with methyltransferase inhibitors, the methylation levels dropped, the BCR-ABL1protein and HOXA9protein levels decreased, but the expression of miR-196b could not be restored, suggesting that decreasing CpG island methylation level of miR-196b promoter enable down-regulation of BCR-ABL1and HOXA9expression. However, whether the effect was caused by changing the expression of miR-196b needed to be further investigated. Clinical samples studies showed that miR-196b expression in CML patients was lower than normal controls, we calculated methylation-positive and methylation-negative ratio of various types of leukemia patients and normal controls, using multi-sample rate χ2to analyze the data, the results showed that the differences between various types of samples were significant (P<0.05), CpG island methylation of miR-196b promoter was significantly higher in CML than in normal (P<0.05). Combining the results of cells and clinical samples, we concluded that: miR-196b was lower in CML, down regulation of miR-196b promoter CpG island methylation level could lead to decreasing of its target genes BCR-ABL1and HOXA9expression, inhiting that miR-196b promoter CpG island hypermethylation may be related with chronic myeloid leukemogenesis. miR-30a expression in CML patients was lower than the normal, cell function studies showed that miR-30a was a tumor suppressor gene in CM, suggesting that low expression of miR-30a was related with chronic myeloid leukemogenesis, but CpG island could not be predicted in miR-30a promoter, suggesting that decreased expression of miR-30a has other reasons, such as a mutation of miR-30a promoter, loss of heterozygosity, the specific mechanism needs to be further studied.In summary, the study on the basis of bioinformatics, used dual luciferase reporter gene assay system to confirm miR-196b and miR-30a target genes initially, followed by gain of function and loss of function study in K562cells to clarify the function of miR-196b and miR-30a in CML, proved that miR-196b target genes including BCR-ABL1and HOXA9, miR-30a target genes including HOXA9, miR-196b and miR-30a played tumor suppressor gene in CML, lastly detected miR-196b promoter CpG island methylation status using MSP and BSP method in45cases of leukemia patients and10normal controls, and examined the expression of miR-196b and miR-30a in CML and normal,results showed that miR-196b was lower in CML than in normal and miR-196b promoter CpG island hypermethylation in CML, suggesting not only miR-196b lower expression may be related with miR-196b promoter CpG island hypermethylation and is one of the mechanisms of chronic myeloid leukemia incidence, but also is a potential target for the treatment of CML. The results also showed that the miR-30a in CML is low expression and plays a tumor inhibitory effect, suggesting that low expression of miR-30a is related with chronic myeloid leukemogenesis, and the specific mechanism needs further study.